Genetic diversity of enterovirus G detected in faecal samples of wild boars in Japan: identification of novel genotypes carrying a papain-like cysteine protease sequence.

The genetic diversity of enterovirus G (EV-G) was investigated in the wild-boar population in Japan. EV-G-specific reverse transcription PCR demonstrated 30 (37.5 %) positives out of 80 faecal samples. Of these, viral protein 1 (VP1) fragments of 20 samples were classified into G1 (3 samples), G4 (1 sample), G6 (2 samples), G8 (4 samples), G11 (1 sample), G12 (7 samples), G14 (1 sample) and G17 (1 sample), among which 11 samples had a papain-like cysteine protease (PL-CP) sequence, believed to be the first discoveries in G1 (2 samples) or G17 (1 sample) wild-boar EV-Gs, and in G8 (2 samples) or G12 (6 samples) EV-Gs from any animals. Sequences of the non-structural protein regions were similar among EV-Gs possessing the PL-CP sequence (PL-CP EV-Gs) regardless of genotype or origin, suggesting the existence of a common ancestor for these strains. Interestingly, for the two G8 and two G12 samples, the genome sequences contained two versions, with or without the PL-CP sequence, together with the homologous 2C/PL-CP and PL-CP/3A junction sequences, which may explain how the recombination and deletion of the PL-CP sequences occured in the PL-CP EV-G genomes. These findings shed light on the genetic plasticity and evolution of EV-G.

Complete genome sequencing and genetic analysis of a Japanese porcine torovirus strain detected in swine feces.

We sequenced the complete genome of a porcine torovirus (PToV) strain from Japan for the first time. Whole-genome analysis revealed that this strain (Iba/2018) has a mosaic sequence composed of at least three genome backgrounds, related to US, Chinese and German PToV strains. Clear recombination breakpoints were detected in the M and HE coding regions. A similarity plot and structural analysis demonstrated that the HE coding region exhibits the highest diversity, and the most sequence variation was found in the lectin domain. PToVs were divided into two lineages in the HE region, whereas clear lineages were not found in other regions.

Phylogenetic analysis of novel posaviruses detected in feces of Japanese pigs with posaviruses and posa-like viruses of vertebrates and invertebrates.

Posaviruses and posa-like viruses are unclassified viruses with sequence similarity to viruses of the order Picornavirales. They have been reported in various vertebrates and invertebrates. We identified 11 posavirus-like sequences in porcine feces and performed phylogenic analysis. Previously reported Japanese posaviruses and those identified in this study clustered with posavirus 1, 4, and 7 and husavirus 1, while five viruses branched into three independent lineages, tentatively named posavirus 10, 11, and 12. Interestingly, posaviruses, except for posavirus 8 and 9, husaviruses, and rasaviruses, formed a cluster consisting of viruses only from pigs, humans, and rats, while posavirus 8 and 9, fisavirus, and basaviruses clustered with posa-like viruses from invertebrates.

Complete genomic analysis and molecular characterization of Japanese porcine sapeloviruses.

The Porcine Sapelovirus (PSV) is an enteric virus of pigs that can cause various disorders. However, there are few reports that describe the molecular characteristics of the PSV genome. In this study, almost the entire genomes of 23 PSVs detected in Japanese pigs were analyzed using bioinformatics. Analysis of the cis-active RNA elements showed that the predicted secondary structures of the internal ribosome entry site in the 5' untranslated region (UTR) and a cis-replication element in the 2C coding region were conserved among PSVs. In contrast, those at the 3' UTR were different for different PSVs; however, tertiary structures between domains were conserved across all PSVs. Phylogenetic analysis of nucleotide sequences of the complete VP1 region showed that PSVs exhibited sequence diversity; however, they could not be grouped into genotypes due to the low bootstrap support of clusters. The insertion and/or deletion patterns in the C-terminal VP1 region were not related to the topology of the VP1 tree. The 3CD phylogenetic tree was topologically different from the VP1 tree, and PSVs from the same country were clustered independently. Recombination analysis revealed that recombination events were found upstream of the P2 region and some recombination breakpoints involved insertions and/or deletions in the C-terminal VP1 region. These findings demonstrate that PSVs show genetic diversity and frequent recombination events, particularly in the region upstream of the P2 region; however, PSVs could currently not be classified into genotypes and conserved genetic structural features of the cis-active RNA elements are observed across all PSVs.

Influence of glucocorticoids on time-of-day-dependent variations in IgE-, histamine-, and platelet-activating factor-mediated systemic anaphylaxis in different mouse strains.

A time-of-day-dependent variation in IgE-mediated passive systemic anaphylaxis was previously reported in ICR mice. In the present study, we investigated time-of-day-dependent variations in IgE-, histamine-, and platelet-activating factor (PAF)-mediated systemic anaphylaxis in C57BL/6, BALB/c, and NC/Nga mice at 9:00 h and 21:00 h, and evaluated the potential influence of glucocorticoids (GCs) on these variations. We found significant time-of-day-dependent variations in IgE-mediated systemic anaphylaxis in C57BL/6 mice, and in histamine- and PAF-mediated systemic anaphylaxis in BALB/c mice. Significant daily variations in IgE-, histamine-, and PAF-mediated systemic anaphylaxis were not observed in NC/Nga mice. Pretreatment with dexamethasone and adrenalectomy abolished the daily variations in IgE-mediated systemic anaphylaxis in C57BL/6 mice and in PAF-mediated systemic anaphylaxis in BALB/c mice, suggesting that GCs from adrenal glands are pivotal in regulating these variations. In contrast, pretreatment with dexamethasone and adrenalectomy did not abolish the daily variation in histamine-mediated systemic anaphylaxis in BALB/c mice, suggesting that GC-independent and adrenal gland-independent mechanisms are important for the variation. The present study demonstrated that time-of-day-dependent variations in systemic anaphylaxis differed among inbred mouse strains and with anaphylaxis-inducing substances. Thus, mouse strains, time of experiment, and anaphylaxis-inducing substances used must be considered to obtain appropriate experimental results.

Complete genome analysis of porcine kobuviruses from the feces of pigs in Japan.

Porcine kobuviruses (PoKoVs) are ubiquitously distributed in pig populations worldwide and are thought to be enteric viruses in swine. Although PoKoVs have been detected in pigs in Japan, no complete genome data for Japanese PoKoVs are available. In the present study, 24 nearly complete or complete sequences of the PoKoV genome obtained from 10 diarrheic feces and 14 non-diarrheic feces of Japanese pigs were analyzed using a metagenomics approach. Japanese PoKoVs shared 85.2-100% identity with the complete coding nucleotide (nt) sequences and the closest relationship of 85.1-98.3% with PoKoVs from other countries. Twenty of 24 Japanese PoKoVs carried a deletion of 90 nt in the 2B coding region. Phylogenetic tree analyses revealed that PoKoVs were not grouped according to their geographical region of origin and the phylogenetic trees of the L, P1, P2, and P3 genetic regions showed topologies different from each other. Similarity plot analysis using strains from a single farm revealed partially different similarity patterns among strains from identical farm origins, suggesting that recombination events had occurred. These results indicate that various PoKoV strains are prevalent and not restricted geographically on pig farms worldwide and the coexistence of multiple strains leads to recombination events of PoKoVs and contributes to the genetic diversity and evolution of PoKoVs.

Characterization and phylogenetic analysis of a novel picornavirus from swine feces in Japan.

During an investigation of porcine fecal viruses using a metagenomics approach, a novel picornavirus was identified from the feces of a healthy two-month-old pig. This virus, named porcine picornavirus Japan (PPVJ), had a standard picornavirus genome organization, including the L protein region. The 5' untranslated region harbored a type II internal ribosomal entry site. This virus was most closely related to lesavirus 1 (amino acid sequence identity: 38.2 %) in P1, equine rhinitis A virus (25.8 %) in P2, and lesavirus 2 (40.9 %) in P3. According to the genus demarcations for the family Picornaviridae (less than 40 %, 40 %, and 50 % amino acid sequence identity in P1, P2, and P3, respectively), PPVJ represents a new genus in the family Picornaviridae. PPVJ was detected in 23.3 % of the fecal samples (from 58.3 % of the farms across a wide area) from pigs less than four months old, by reverse transcription PCR, using specific primers designed from the 3D sequence, followed by sequencing. The host range and pathogenic potential of this virus in animals is yet to be determined.

Identification of further diversity among posaviruses.

Recently, there have been reports of new members of posavirus-like viruses in the order Picornavirales. In this study, using a metagenomics approach, 11 posavirus-like sequences (>7,000 nucleotides) were detected in 155 porcine fecal samples. Phylogenetic analysis revealed that the newly identified virus sequences, together with other posavirus-like viruses, form distinct clusters within the order Picornavirales, composed of eight genogroups and unassigned sequences based on amino acid sequences of the helicase and RNA-dependent RNA polymerase regions, with <40 % and <50 % sequence identity, respectively. We propose further classifications of highly diverse posavirus populations based on newly identified sequences from Japanese pig feces.

Development of one-step real-time reverse transcriptase-PCR-based assays for the rapid and simultaneous detection of four viruses causing porcine diarrhea.

Porcine diarrhea caused by viruses is a major problem of the pig farming industry and can result in substantial losses of revenue. Thus, diagnosing the infectious agents is important to prevent and control diseases in pigs. We developed novel one-step real-time quantitative RT-PCR (qPCR) assays that can detect four porcine diarrheal viruses simultaneously: porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine group A rotavirus (PRVA). The qPCR analysis takes only 75 minutes to detect the presence of the four viruses. The limits of detection of our new assays for PEDV, TGEV, PDCoV, and PRVA were 100, 10, 10 and 10 copies per reaction, respectively. The sensitivity of qPCR was 1-1000 times higher than that of published gel-based RT-PCR. We used our qPCR method to successfully diagnose clinical samples from infected pigs, and no false positive results were obtained. In conclusion, qPCR can drastically reduce the diagnostic time to detect viruses compared to currently employed methods. We predict that the qPCR assays will become a useful tool for detecting viral infections that cause diarrhea and other complications in pigs.

Whole-genome sequence analysis of G3 and G14 equine group A rotaviruses isolated in the late 1990s and 2009-2010.

Equine group A rotavirus (RVA) G3P[12] and G14P[12] strains cause gastroenteritis in foals worldwide. Both of these strains have been co-circulating in Japan since G14P[12] strains emerged in the late 1990s. Although it is important to comprehensively understand the evolution of RVA strains, whole-genome sequence data on recent equine RVA strains in Japan are lacking. Therefore, in this study, whole-genome analysis of 23 equine RVA isolates from the late 1990s and 2009-2010 and the vaccine strain RVA/Horse-tc/JPN/HO-5/1982/G3P[12] (HO-5) was performed. The G3 strains, including strain HO-5, shared a G3-P[12]-I6-R2-C2-M3-A10-N2-T3-E2-H7 genotype constellation, and all of their 11 gene segments were highly conserved, regardless of the year of isolation. G14 strains also exhibited an identical genotype constellation (G14-P[12]-I2-R2-C2-M3-A10-N2-T3-E2-H7), but, phylogenetically, segregated into two lineages within the VP7-G14 and NSP4-E2 genotypes. G14 strains were closely related to G3 strains in their VP4, VP1-3, NSP1-3 and NSP5 gene segments. Interestingly, the NSP4 gene of all G3 and G14 strains isolated in the late 1990s branched into a bovine-RVA-like NSP4 gene cluster. These results indicate that, apart from VP7, VP6, and NSP4 genes, the Japanese equine RVA strains share a highly conserved genetic backbone, and that strains possessing a bovine-RVA-like NSP4 gene were predominant in the late 1990s in Japan.

Full genome analysis of bovine astrovirus from fecal samples of cattle in Japan: identification of possible interspecies transmission of bovine astrovirus.

A viral metagenomics approach was used to investigate fecal samples of Japanese calves with and without diarrhea. Of the different viral pathogens detected, read counts gave nearly complete astrovirus-related RNA sequences in 15 of the 146 fecal samples collected in three distinct areas (Hokkaido, Ishikawa, and Kagoshima Prefectures) between 2009 and 2015. Due to the lack of genetic information about bovine astroviruses (BoAstVs) in Japan, these sequences were analyzed in this study. Nine of the 15 Japanese BoAstVs were closely related to Chinese BoAstVs and clustered into a lineage (tentatively named lineage 1) in all phylogenetic trees. Three of 15 strains were phylogenetically separate from lineage 1, showing low sequence identities, and clustered instead with an American strain isolated from cattle with respiratory disease (tentatively named lineage 2). Interestingly, two of 15 strains clustered with lineage 1 in the open reading frame (ORF)1a and ORF1b regions, while they clustered with lineage 2 in the ORF2 region. Remarkably, one of 15 strains exhibited low amino acid sequence similarity to other BoAstVs and was clustered separately with porcine astrovirus type 5 in all trees, and ovine astrovirus in the ORF2 region, suggesting past interspecies transmission.

Effects of a selective casein kinase 1δ and ε inhibitor on FcεRI expression and IgE-mediated immediate-type cutaneous reactions in dogs.

The molecular clock network in mast cells has been shown to be a factor responsible for circadian regulation of allergic inflammation. PF670462 is a selective inhibitor of casein kinase 1δ and ε (CK1δ/ε) that control the posttranslational modification of clock proteins. The aims of this study were to evaluate the effects of PF670462 on gene and protein expression of FcεRI, the high-affinity IgE receptor, in canine mast cells and on IgE-mediated immediate-type cutaneous reactions in dogs. PF670462 decreased mRNA expression of FcεRIα and ß, but not γ, and protein expression of FcεRI in a canine mast cell line. Furthermore, PF670462 suppressed IgE-mediated immediate-type cutaneous erythema in dogs. These findings indicate that CK1δ/ε function as regulators for FcεRI expression and IgE-mediated cutaneous reactions in dogs.

Complete genome sequencing and genetic characterization of porcine sapovirus genogroup (G) X and GXI: GVI, GVII, GX, and GXI sapoviruses share common genomic features and form a unique porcine SaV clade.

Sapoviruses (SaVs) are enteric viruses belonging to the family Caliciviridae that infect humans and animals, including pigs. To date, SaVs have been classified into 19 genogroups (G) based on complete VP1 sequences; however, complete genome sequences of some SaV Gs are not yet available. In this study, we determined the full genome sequences of four SaVs (two GX and two GXI SaVs) and analyzed them together with those of other SaVs. The complete genome sequences of GX and GXI SaVs, excluding the poly(A) tails, were 7124, 7142, 7170, and 7179 nucleotides, which were shorter than those of other SaVs, except for porcine GVI and GVII viruses. Genetic characterization revealed that GX SaVs and GXI SaVs shared common features with GVI and GVII viruses, such as the first 10 amino acid residues in the ORF1 coding region, a shorter ORF1 than that of the other genogroups, and the predicted secondary structure of the 5' end of the genome and the starting region of non-structural protein/structural protein junction. Phylogenetic analyses showed that GX and GXI SaVs branched with porcine GVI, GVII, and GIX SaVs and formed a clade consisting of only porcine SaVs. These findings suggest that porcine GX and GXI SaVs together with porcine GVI, GVII, and possibly GIX SaVs, evolved from a common ancestor in the porcine population.

First identification of Sapoviruses in wild boar.

Sapoviruses (SaVs) are enteric viruses that have been detected in human and animals previously; however, SaVs have not been identified in wild boar yet. Using a metagenomics approach, we identified SaVs in fecal samples of free-living wild boars in Japan for the first time. Six of the 48 specimens identified belonged to one genogroup (G)III, one GV and four GVI SaV sequence reads. We successfully determined complete genome of GV and GVI SaV strains using the long reverse transcription PCR strategy and the 5' rapid amplification of cDNA end method. Phylogenetic tree analysis and pairwise distance calculation revealed that GV SaV detected from wild boar was related to recently assigned GV.5 strains from pig, while GVI SaV was assigned to a new genotype within GVI. Moreover, wild boar may act as a reservoir for transmission of SaVs to the pig population (and vice versa) because GIII, GV, and GVI SaVs were all detected in pigs previously.

Whole genome analysis of a novel picornavirus related to the Enterovirus/Sapelovirus supergroup from porcine feces in Japan.

A novel virus related to the Enterovirus/Sapelovirus supergroup in the family Picornaviridae was identified in healthy porcine feces in Japan by using a metagenomics approach. The genome of the virus, named Sapelo-like porcine picornavirus Japan (SPPVJ) Pig/Isi-Im1/JPN/2016, had a type-IV internal ribosomal entry site and carried a 6978-nucleotide-long single open reading frame encoding a 2326 amino acids (aa) polyprotein precursor. The coding sequence region consisted of leader protein (68 aa), a structural protein region P1 (824 aa), and the non-structural protein regions P2 (672 aa) and P3 (762 aa). Among representative picornaviruses, the P1, 2C, and 3CD regions of SPPVJ had the highest aa identities of 64.4%, 61.9%, and 73.3%, respectively, with the corresponding regions of sapelo-like bat picornavirus BtVs-PicoV/SC2013. Sequencing analysis of the RT-PCR products derived from the 5' untranslated and 3D regions revealed the presence of SPPVJ in 17.8% (19/107) of the feces from healthy and diarrheal pigs in 12 farms in 2015-2016. Further studies are needed to determine the origin and pathogenic potential of SPPJV in pigs and other mammals.

Effect of interleukin-1ß on occludin mRNA expression in the duodenal and colonic mucosa of dogs with inflammatory bowel disease.

BACKGROUND: Mucosal imbalance of interleukin (IL)-1ß and IL-1 receptor antagonist (Ra) has been reported in the duodenal mucosa of dogs with inflammatory bowel disease (IBD). However, the imbalance in the colonic mucosa and its role in duodenitis and colitis in IBD of dogs remain unclear. OBJECTIVES: To measure the expression of IL-1ß and IL-1Ra proteins in the colonic mucosa of dogs with IBD, and to determine the effect of IL-1ß on expression of occludin (ocln) mRNA, a tight junction component, in the duodenal and colonic mucosa of dogs with IBD. ANIMALS: Twelve dogs with IBD and 6 healthy dogs. METHODS: IL-1ß and IL-1 Ra proteins in the colonic mucosa were quantified by ELISA in 7 of the 12 dogs with IBD. Expression of ocln mRNA in the duodenal and colonic mucosa was examined in the 12 dogs by real-time PCR. RESULTS: The ratio of IL-1ß to IL-1Ra in the colonic mucosa was significantly higher in dogs with IBD than in healthy dogs. The ex vivo experiment determined that IL-1ß suppressed expression of ocln mRNA in the colonic mucosa, but not in the duodenal mucosa, of healthy dogs. Expression of ocln mRNA in the colonic mucosa, but not in the duodenal mucosa, was significantly lower in dogs with IBD than in healthy dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: A relative increase in IL-1ß may attenuate ocln expression, leading to intestinal barrier dysfunction and promotion of intestinal inflammation in the colonic mucosa, but not in the duodenal mucosa, of dogs with IBD.

Genetic diversity and recombination of enterovirus G strains in Japanese pigs: High prevalence of strains carrying a papain-like cysteine protease sequence in the enterovirus G population.

To study the genetic diversity of enterovirus G (EV-G) among Japanese pigs, metagenomics sequencing was performed on fecal samples from pigs with or without diarrhea, collected between 2014 and 2016. Fifty-nine EV-G sequences, which were >5,000 nucleotides long, were obtained. By complete VP1 sequence analysis, Japanese EV-G isolates were classified into G1 (17 strains), G2 (four strains), G3 (22 strains), G4 (two strains), G6 (two strains), G9 (six strains), G10 (five strains), and a new genotype (one strain). Remarkably, 16 G1 and one G2 strain identified in diarrheic (23.5%; four strains) or normal (76.5%; 13 strains) fecal samples possessed a papain-like cysteine protease (PL-CP) sequence, which was recently found in the USA and Belgium in the EV-G genome, at the 2C-3A junction site. This paper presents the first report of the high prevalence of viruses carrying PL-CP in the EV-G population. Furthermore, possible inter- and intragenotype recombination events were found among EV-G strains, including G1-PL-CP strains. Our findings may advance the understanding of the molecular epidemiology and genetic evolution of EV-Gs.

Continuous large-scale production of the cytokine CXCL8 from a novel porcine cell line.

Cytokine production from two unstimulated porcine cell lines (SL-24 and SK-L) was examined using porcine cytokine detection ELISA kits and RT-PCR. Porcine IL-1 alpha, IL-6, and CXCL8 were detected in all samples examined. In particular, the SL-24 cell line (derived from bone marrow cells of a malignant lymphoma-affected pig), produced large amounts of porcine CXCL8. Flow cytometer analysis showed the cell line to be strongly CD44 positive, and was therefore considered to be of monocyte or macrophage origin. Porcine CXCL8 production was greatest (83.86 +/- 32.33 ng/mL) at six days post-cultivation. The SK-L cell line (derived from porcine kidney) also produced CXCL8, but production was less than 1.5 ng/mL. Porcine CXCL8 from the SL-24 cell line, induced chemotactic activity in porcine neutrophils, while the production of CXCL8 from the SL-24 cell line was inhibited by dexamethasone, which suggests that the mechanism of CXCL8 production is related to an NF-kappaB binding site. The production of CXCL8 from the SL-24 cell line was enhanced by the addition of recombinant porcine IL-15, which is the first reported observation of such CXCL8 production. Cloning of the SL-24 cell line by limited dilution revealed two types of cells present in the starting population. One cell type, designated as long-form cells (LC), produced large amounts of CXCL8, while the other, designated short-form cells (SC), produced small amounts of the cytokine. The LC cells were adapted to grow in serum-free medium in which they produced large amounts of CXCL8. The large-scale production of porcine CXCL8 from the SL-24 cell line will be of value in determining the mechanism of cytokine production and as a source of naturally produced porcine CXCL8.

Generation of recombinant bovine interferon tau in the human embryonic kidney cell line and its biological activity.

The objective of this study was to generate recombinant bovine interferon tau (rbIFNT) in mammalian hosts. The complementary DNA encoding bovine IFNT2 was cloned for the construction of pRcRSV-bIFNT2 expression vector. The expression vector was transfected to 293 cells. Transfected cells harboring expression vector were selected with G418. Highly expressing clonal line was adapted to serum-free suspension culture in a spinner flask. The recombinant protein had 24 kDa apparent molecular mass, suggesting being expressed as a glycoprotein, and was purified from serum-free conditioned medium by the combination of Diethylaminoethanol Sepharose ion exchange and Sephacryl S-200 HR gel filtration. A total of 7.3 mg rbIFNT was obtained from 13.5 L conditioned medium. Generated rbIFNT was biologically active in terms of antiviral activity measured by the plaque inhibition assay with Madin-Darby bovine kidney cells and the vesicular stomatitis virus. The recombinant protein was also utilized for immunization to raise antibodies in the rabbit. The generated antibody was capable of use in both Western blotting and the binding assay. The results in the present study suggest that a certain amount of rbIFNT is raised in mammalian hosts by using conventional plasmid vector and its antibody provides useful tools for studies in the biology of bovine IFNT.

Expression of epithelial cell-derived cytokine genes in the duodenal and colonic mucosae of dogs with chronic enteropathy.

It remains unclear whether epithelial cell-derived cytokines, including interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP), contribute to development of canine chronic enteropathy (CE), which includes antibiotic-responsive enteropathy (ARE), food-responsive enteropathy (FRE) and inflammatory bowel disease (IBD). In the present study, we examined mRNA expression of il-25, il-33 and tslp in the duodenal and colonic mucosae of dogs with ARE, FRE and IBD. Real-time PCR analysis revealed that mRNA expression of il-33 was significantly lower in the duodenum in dogs with FRE than in healthy dogs. The results suggest that epithelial cell-derived cytokines may not be an inducer of Th2-type immunity in the gut of dogs with CE, and decreased expression of IL-33 may be involved in induction of FRE. Further studies are required to clarify roles of epithelial cell-derived cytokines, especially IL-33, in the pathogenesis of canine CE.